Process for improving glucose metabolism, satiety, and nutrient absorption in companion animals

ABSTRACT

A process for feeding an animal a diet which alters the function and morphology of the gastrointestinal tract (GIT), a large lymphoid organ in the animal and which improves glucose metabolism, satiety, and nutrient absorption. The process involves feeding a companion animal such as, for example, a dog or cat a diet of a pet food composition containing fermentable fibers which have an organic matter disappearance (OMD) of 15 to 60 percent when fermented by fecal bacteria for a 24 hour period, the fibers being present in amounts from about 1 to 11 weight percent of supplemental total dietary fiber. The animal is maintained on the diet for a sufficient period of time to allow the fermentable fibers to ferment in the GIT of the animal.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This patent application is a continuation of U.S. patentapplication Ser. No. 09/723,163, filed Nov. 27, 2000, which is acontinuation of U.S. patent application Ser. No. 09/055,790, filed Apr.6, 1998, which claims the benefit of U.S. Provisional Patent ApplicationSerial No. 60/042,957, filed Apr. 7, 1997.

BACKGROUND OF THE INVENTION

[0002] This invention relates to a process involving the use of a petfood composition containing fermentable fibers to improve glucosemetabolism, satiety, and nutrient absorption in companion animals suchas, for example, dogs and cats.

[0003] Recent research has suggested that dietary fiber is important forits fermentation properties in the large intestine of dogs and cats. Forexample, Reinhart, U.S. Pat. No. 5,616,569, describes the addition offermentable dietary fiber to a pet food composition for the purpose ofmaintaining normal gastrointestinal function and ameliorating chronicdiarrhea in animals. Howard et al FASEB J. (1996) 10:A257, teach thatfermentable fiber consumption by dogs can result in the partition ofwaste nitrogen from the urine to the feces, increasing nitrogenexcretion through the feces of the animal. Sunvold et al, J. Anim. Sci.(1995) 73:1099-1109, found that feeding moderately fermentable fibers todogs could promote gastrointestinal tract health by optimizing shortchain fatty acid (SCFA) production in the intestines of the animals.

[0004] Certain animals, such as dogs, as well as humans, sometimessuffer from diabetes or have an impaired ability to regulate blood sugarlevels. There are many causes of diabetes. Where diabetes or impairedblood glucose regulation has been diagnosed, medication and diet for theanimal should be closely controlled. Currently, diets having highconcentrations of nonfermentable fibers are used to treat diabetes.However, these nonfermentable fiber-containing diets often impairnutrient absorption by the animal, resulting in undesirable effects onthe animal's health and well being.

[0005] Certain animals also may have a tendency towards excess caloricintake which increases the risk of the animal developing diabetes orother chronic diseases. It would be desirable to be able to managecaloric intake through dietary means so that the animal would becomesated after meals, but without excessive caloric intake.

[0006] Other animals may have difficulty in digesting and absorbingnutrients from their diets. For example, animals which exhibit exocrinepancreatic insufficiency (EPI), a condition in which there is aninsufficient secretion of enzymes by the pancreas, struggle to digestnutrients normally, especially fats, in their diets. It would bedesirable to be able to improve such animals' nutrient absorptioncapabilities. Thus, there remains a need for additional dietary measureswhich will improve glucose metabolism, satiety, and nutrient absorptionin companion animals without the adverse effects of diets containingnonfermentable fibers.

SUMMARY OF THE INVENTION

[0007] The present invention meets that need by providing a process forfeeding an animal a diet which alters the function and morphology of thegastrointestinal tract (GIT), a large lymphoid organ, in ways which arebeneficial to the animal's health and well being. The process involvesfeeding a companion animal such as, for example, a dog or cat a diet ofa pet food composition containing fermentable fibers which have anorganic matter disappearance (OMD) of 15 to 60 percent when fermented byfecal bacteria for a 24 hour period, the fibers being present in amountsfrom about 1 to 11 weight percent of supplemental total dietary fiber.The animal is maintained on the diet for a sufficient period of time toallow the fermentable fibers to ferment in the GIT of the animal. Thisfermentation results in an upregulation in the secretion of GLP-1 whichimproves glucose homeostasis and promotes satiety in the animal. Thediet also enhances the absorption of nutrients by the animal byincreasing the transport of D-glucose and lauric acid which areindicators of carbohydrate and fat absorption, respectively.

[0008] Preferably, the pet food composition contains from 2 to 10 weightpercent of supplemental total dietary fiber of fermentable fibers. Morepreferably, the pet food composition contains from 3 to 9 weight percentof supplemental total dietary fiber of fermentable fibers. Mostpreferably, the pet food composition contains from 4 to 7 weight percentof supplemental total dietary fiber of fermentable fibers.

[0009] Preferably, the fermentable fibers have an organic matterdisappearance of 20 to 50 percent. More preferably, the fermentablefibers have an organic matter disappearance of 30 to 40 percent.

[0010] In addition, the fermentable fibers are preferably selected fromthe group consisting of beet pulp, gum arabic, gum talha (a form of gumarabic), psyllium rice bran, carob bean gum, citrus pulp, pectin,fructooligosaccharides or inulin, mannanoligosaccharides and mixturesthereof. More preferably, the fermentable fibers are selected from thegroup consisting of beet pulp, gum arabic and fructooligosaccharides.Most preferably, the fermentable fibers are a blend of beet pulp, gumtalha, and fructooligosaccharides. A preferred weight ratio of beet pulpto fructooligosaccharides in the fermentable fiber blend is from about3:1 to 6:1, and most preferably 4:1. A preferred weight ratio of beetpulp to gum talha to fructooligosaccharide is 6:2:1.5.

[0011] Accordingly, it is a feature of the present invention to providea pet food composition and process for altering the function andmorphology of the gastrointestinal tract to improve glucose metabolismand enhance glucose homeostasis, improve satiety, and enhance nutrientabsorption in an animal. This, and other features and advantages of thepresent invention, will become apparent from the following detaileddescription, the accompanying drawings, and the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIGS. 1A-1C illustrate the effect of fermentable fiber on plasmaGLP-1 (A), insulin (B), and glucose concentrations (C) afteradministration of an oral glucose tolerance test (OGTT), withsignificantly different time points (p<0.05) indicated by “*”;

[0013] FIGS. 2A-2C illustrate the incremental area under the curve forplasma GLP-1 (A), insulin (B), and glucose (C) after administration ofan oral glucose tolerance test (OGTT);

[0014]FIG. 3 is a chart showing the effect of fermentable fiber onintestinal proglucagon mRNA;

[0015] FIGS. 4A-4B are charts showing the effect of fermentable fiber onvilli height (A) and crypt depth (B) in canine intestinal sections;

[0016] FIGS. 5A-5B illustrate the effect of fermentable fiber on the invitro uptake of glucose into the jejunum (A) and ileum (B) of dogs;

[0017]FIG. 6 is a chart of the effect of fermentable fiber on intestinalSGLT-1 transporter mRNA;

[0018] FIGS. 7A-7B illustrate the effect of fermentable fiber on jejunal(A) and ileal (B) SGLT-1 transporter abundance in dogs;

[0019] FIGS. 8A-8B illustrate the effect of fermentable fiber onintestinal GLUT2 transporter abundance in jejunum (A) and ileum (B) indogs;

[0020]FIG. 9 is a graph of the rates of glucose (GLC) and proline (PRO)uptake in the proximal (P), mid (M), and distal (D) intestine;

[0021]FIG. 10 illustrates the uptake by the proximal intestine as afunction of glucose concentration;

[0022]FIG. 11 illustrates the uptake by the proximal intestine as afunction of proline concentration; and

[0023]FIG. 12 is a chart illustrating the intestinal capacities of dogsto absorb glucose (GLC) and proline (PRO) in the proximal (P), mid (M),and distal (D) intestine.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] The present invention uses a pet food composition containingfermentable fibers to alter the function and morphology of thegastrointestinal tract of the animal. This provides a number of benefitsto the animal. First, glucose metabolism is improved and glucosehomeostasis is enhanced in the animal. While not wishing to be bound byany particular theory, it is believed that the improvement in glucoseregulation in the animals results at least in part from the increasedlevels of insulinotropic gut hormones such as GLP-1 which are secretedin the gastrointestinal tract. GLP-1 is a potent insulinotropic hormoneand potential antidiabetogenic agent. This upregulation of GLP-1 isbelieved to increase intestinal glucose transport capacity and improveglucose homeostasis in the animal. Increased levels of GLP-1 in the GITof the animal also improve satiety in the animal and reduce the animal'stendency to overeat. These results are surprising in view of the priorart practice of using very high fiber concentration in animal diets, butusing low fermentablility fibers such as cellulose to attempt toaccomplish this result.

[0025] Further, the presence of fermentable fibers in the diet increasesthe transport of D-glucose and lauric acid in the jejunum (mid portion)of the small intestine. D-glucose and lauric acid are indicators ofcarbohydrate and fat absorption, respectively, in an animal. Thus,healthy animals will benefit from the process of the present inventionwhich improves nutrient absorption. However, animals which are sufferingfrom certain disease states such as exocrine pancreatic insufficiency(EPI) will benefit even more. EPI results from insufficient secretion ofenzymes by the pancreas, with such enzymes being needed by the animalfor normal nutrient digestion. Animals with EPI struggle to digestdietary nutrients, especially fats. Animals with EPI which are fed thepet food composition of this invention will benefit by an improvedability to absorb dietary nutrients.

[0026] The present invention uses a pet food composition containingfermentable fibers which display certain organic matter disappearancepercentages. The fermentable fibers used in the present invention havean organic matter disappearance (OM D) of from about 15 to 60 percentwhen fermented by fecal bacteria in vitro for a 24 hour period. That is,from about 15 to 60 percent of the total organic matter originallypresent is fermented and converted by the fecal bacteria. The organicmatter disappearance of the fibers is preferably 20 to 50 percent, andmost preferably is 30 to 40 percent.

[0027] Thus, in vitro OMD percentage may be calculated as follows:

{1-[(OM residue−OM blank)/OM initial]}×100,

[0028] where OM residue is the organic matter recovered after 24 hoursof fermentation, OM blank is the organic matter recovered incorresponding blank tubes (i.e., tubes containing medium and dilutedfeces, but no substrate), and OM initial is that organic matter placedinto the tube prior to fermentation. Additional details of the procedureare found in Sunvold et al, J. Anim. Sci. 1995, vol. 73:1099-1109.

[0029] The pet food composition can be any suitable pet food formulawhich also provides adequate nutrition for the animal. For example, atypical canine diet for use in the present invention may contain about30% crude protein, about 20% fat, and about 10% total dietary fiber.However, no specific ratios or percentages of these or other nutrientsare required.

[0030] Fermentable fibers which are useful in the present inventionproduce short chain fatty acids (SCFAs) within a range of from about 28to about 85 mmol SCFA per 1000 Calories (kcals) of metabolizable energy(ME), and more preferably within a range of from about 42 to about 71mmol SCFA per 1000 ME kcals. This equates to a composition which has atotal fermentable fiber content which yields from about 100 to about 350mmol SCFA/kg of diet.

[0031] Millimoles of SCFAs per 1000 metabolizable energy kilocaloriesare calculated by first calculating the total Calories of metabolizableenergy (ME) in a given diet composition per kilogram of the composition.The number of grams per 1000 kcal ME may be derived from the firstcalculation. Then the grams, and thus millimoles, of the fermentablefiber components of the composition may be calculated.

[0032] The fermentable fiber of the present invention may be any fibersource which intestinal bacteria present in the animal can ferment toproduce significant quantities of SCFAs. “Significant quantities” ofSCFAs, for purposes of this invention, are amounts over 0.5 mmol oftotal SCFAs/gram of substrate in a 24 hour period. Preferred fibersinclude beet pulp, gum arabic (including gum talha), psyllium, ricebran, carob bean gum, citrus pulp, pectin, fructooligosaccharides orinulin, mannanoligosaccharides and mixtures of these fibers. Morepreferably, the fermentable fibers are selected from the groupconsisting of beet pulp, gum arabic and fructooligosaccharides. Mostpreferably, the fermentable fibers are a blend of beet pulp, gum talha,and fructooligosaccharides. A preferred weight ratio of beet pulp tofructooligosaccharides in the fermentable fiber blend is from about 3:1to 6:1, and most preferably 4:1. A preferred weight ratio of beet pulpto gum talha to fructooligosaccharide is 6:2:1.5.

[0033] The fermentable fibers are used in the pet food composition inamounts from 1 to 11 weight percent of supplemental total dietary fiber,preferably from 2 to 10 weight percent, and most preferably from 3 to 7weight percent.

[0034] A definition of “supplemental total dietary fiber” first requiresan explanation of “total dietary fiber”. “Total dietary fiber” isdefined as the residue of plant food which is resistant to hydrolysis byanimal digestive enzymes. The main components of total dietary fiber arecellulose, hemicellulose, pectin, lignin and gums (as opposed to “crudefiber”, which only contains some forms of cellulose and lignin).“Supplemental total dietary fiber” is that dietary fiber which is addedto a food product above and beyond any dietary fiber naturally presentin other components of the food product. Also, a “fiber source” isconsidered such when it consists predominantly of fiber.

[0035] In order that the invention may be more readily understood,reference is made to the following examples which are intended toillustrate the invention, but not limit the scope thereof.

EXAMPLE 1

[0036] Diets, see Table 1, were formulated to be isonitrogenous andisoenergetic and to provide approximately 19.5 MJ/kg diet with 35% ofthe energy from carbohydrate, 30% from fat and 35% from protein. The lowfermentable fiber (LFF) diet contained wood cellulose as the fibersource and the high fermentable fiber diet (HFF) diet contained a blendof more fermentable plant fibers (beet pulp, Michigan Sugar, Saginaw,Mich.; gum arabic, TIC Gums, Belcamp, Md.; fructooligosaccharides (FOS),Golden Technologies Corporation, Golden, Colo.). The ratio of beet pulpto gum arabic to FOS was about 6:2:1.5. The ratio of beet pulp to FOSwas about 4:1.

[0037] Adult mongrel dogs (n=16) were utilized. Upon arrival, animalswere acclimatized for 7 days and fed a nutritionally complete diet(Can-Pro, Beaumont, AB). All dogs were weighed daily and individuallyfed to meet energy requirements using the formula: Energy intake(MJ)=0.553×kg (body weight)^(0.67). Food was offered once daily between0900-1000 hours and water was available ad libitum. A crossoverexperimental design was used whereby dogs were randomly assigned toreceive the HFF or LFF diet for 14 days, followed by the alternate dietfor an additional 14 days. Because the 16 dogs could not be accommodatedat one time, the dogs were paired throughout the experiment.

[0038] Oral Glucose Tolerance Test. Food was removed at 1600 hours ondays 13 and 27. At 0845-0900 hours on days 14 and 28, the dogs wereloosely restrained in a table sling and an oral glucose tolerance test(OGTT) was conducted using 70% (w/w) dextrose to provide 2 g glucose/kgbody weight. Peripheral blood was sampled at 0, 15, 30, 45, 60, 90 and120 min via an Insyte-W 20GA 2″ catheter (Becton-Dickinson VascularAccess, Sandy, Utah) placed in the saphenous vein.

[0039] Peripheral blood samples. Blood samples for general chemistryscreen and complete blood counts (2 ml) were placed into 3 mlheparinized Vacutainer tubes (trademark, Becton-Dickinson, Sunnyvale,Calif.) and stored on ice until analysis. Hematological analyses wereconducted using a Coulter STKS instrument (Courter Electronics Inc.,Hialeah, Fla.) and manual differential counts were performed. Bloodsamples for insulin and GLP-1 analysis were collected into 10 ml EDTAheparinized Vacutainer tubes with aprotinin (500 KIU/ml blood, SigmaChemicals, St. Louis Mo.) and stored at −70° C. (GLP-1) or −35° C.(insulin). Blood samples for serum glucose determinations were placed in250 μL microcentrifuge tubes and centrifuged at 2900×g for 10 min atroom temperature. The serum was removed by pipes and stored at −35° C.

[0040] Intestinal samples. On day 28, the dogs were anesthetized byintravenous injection of somnitol (MTC Pharmaceuticals, Cambridge, ON)using 1 ml/2.27 kg body weight via the saphenous catheter subsequent tothe OGTT. Intestinal samples were taken for northern blot analysis andimmediately placed in liquid nitrogen. Jejunal and ileal samples fornutrient uptake assays were placed in ice-cold saline and assays wereperformed within 30 min of sampling. Jejunal and ileal segments werescraped to obtain mucosal samples for western blot analyses.Histological samples were placed directly into formalin and slides wereprepared.

[0041] Glucose. Serum glucose was determined using the Sigma DiagnosticsGlucose (Trinder) Reagent for the enzymatic determination of glucose at505 nm (Cat #315-100, Sigma Chemical, St. Louis Mo.).

[0042] Insulin. Serum insulin concentrations were determined using theCoat-A-Count® I¹²⁵ diagnostic radioimmunoassay (Cat # TKIN1 ,Diagnostics Products Corporation, Los Angeles Calif.).

[0043] Plasma GLP-1 (7-36)NH₂ Extraction. GLP-1 immunoreactive peptideswere extracted as from 2.5 ml of plasma as described by Reimer andMcBurney, Endocrinol. 137:3948-3956 (1996). A SEP-COLUMN was usedcontaining 200 mg of C₁₈ (Cat # RIK-SEPCOL 1, Peninsula-Laboratories,Belmont, Calif.) with Buffer A (0.1% trifluoroacetic acid (Cat #RIK-BA-1) and Buffer B (60% acetonitrile (Cat # RIK-BB 1) as elusionsolvents. Samples were lyophilized overnight using a Speed-Vac(trademark, Savant Inc., Midland, Mich.) and stored at −70° C.

[0044] Intestinal GLP-1(7-36)NH₂ Extraction. Extraction ofGLP-1(7-36)NH₂ from intestinal segments has been described by Xioyan,PhD thesis, University of British Columbia, Vancouver (1996) and wascarried out with modifications. 400-500 mg of each segment (jejunum,ileum and colon) was added to a 12×75 mm Simport polypropylene tube(Fischer Scientific, Edmonton, AB) with 0.5 ml 2M acetic acid and boiledfor 1 hour. Tubes were centrifuged at 4500×g for 10 min, the supernatantcollected, transferred to a fresh tube and neutralized with 1N NaOH. ForRIA purposes, the sample of supernatant was diluted 1:10 with RIA buffer(100 mM Tris, 50 mM NaCl, 200 mM Na₂-EDTA, 0.2 g/L Na azide, pH 8.5) togive a final sample volume of 100 μL.

[0045] GLP-1(7-36)NH₂ Radioimmunoassay. Concentrations of GLP-1(7-36)NH₂ were measured using a competitive binding radioimmunoassaydescribed by Xiaoyan (1996) with modifications. The lyophilized plasmasamples were reconstituted in 250 μL of RIA assay buffer (100 mM Tris,50 mM NaCl, 20 mM Na₂-EDTA, 0.2 g/L Na azide, pH 8.5). Polypropylenetubes (12 mm×75 mm) were used for controls, standards and samples andthe entire procedure was carried out on ice. GLP-1 (7-36 NH₂) standards(Peninsula Laboratories, Belmont, Calif.) made from serial dilutions,ranged from 4000 pg/ml to 15 pg/ml. Tubes were mixed and incubated 24hours at 4° C. Following incubation, 50 Bq of ¹²⁵I-GLP-1 (736)NH₂ tracerwas added to the tubes, the tubes were mixed by vortexing and incubatedfor 48 hours at 4° C. A dextran-charcoal suspension (4 g/L dextran T70,80 g/L charcoal in assay buffer) was added to all tubes (100 μL) exceptTC tubes. Tubes were mixed by vortexing and left on ice for 15 min,centrifuged at 2200×g for 30 min and 600 μL of supernatant wastransferred to new tubes which were counted using a Cobra™ Auto-Gammacounter (Packard Instrument Company, Downers Grove, Ill.).

[0046] GLP-1(7-36)NH₂ Iodination. GLP-1(7-36 NH₂) was iodinated usingthe chloramine-T method as described by Xiaoyan (1996). The cartridgewas primed by allowing 10 ml acetonitrile with 0.1% trifluoroacetic acid(TFA) followed by 10 ml of ddH₂O with 0.1% TFA to flow through. Thecartridge was dried by pushing 10 ml air through the cartridge with asyringe. The iodination was carried out by first dissolving 30-40 μg ofGLP-1 in 30-40 μL of ddH₂O, then 10 μL was transferred to a fresheppendorf tube. To this, 10 μL 0.5 M P0₄ (pH 7.0) was added followed by0.5 m Ci ¹²⁵I. Chloramine-T (10 μL) was added and the tube was tappedfor exactly 30 seconds. Sodium metabisulfite (5 mg/ml) was added,followed by 1 ml of 0.1% TFA which was then transferred to the primedcolumn. Gentle pressure was applied to the column using a 10 cc syringe.Acetonitrile with 0.1% TFA was used as the elutant to acquire 5fractions. Acetonitrile (5 ml, 10%+0.1% TFA) and acetonitrile (5 ml,20%+0.1% TFA) are the first 2 elutants used in that order and thefractions were collected into 14 mL round bottom tubes. Then 30%acetonitrile (1 ml+0.1% TFA, 4 times), 38% acetonitrile (1 ml+0.1% TFA,once) and 40% acetonitrile (1 ml+0.1% TFA 5 times) were used as the nextelutants in that order and the fractions were collected in smallpolypropylene tubes. Each eluted fraction was mixed well and 10 μL fromeach fraction was counted using a Cobra™ Auto-Gamma counter. The labelwas usually eluted in fraction 1, 2 and/or 3 of the 40% acetonitrile.Fractions containing the labeled GLP-1(7-36)NH₂ were pooled and storedat −35° C. The ¹²⁵IGLP-1 (7-36)NH₂ has a storage life of approximately 2weeks.

[0047] Isolation of Total RNA. Total RNA was isolated from eachintestinal segment using Trizol™ (Gibco BRL, Burlington, ON) accordingto the protocol provided by the manufacturer. 400-500 mg of tissue wasground in a pre-chilled sterile mortar with pestle. The ground tissue(200 mg in duplicate) was weighed and transferred in duplicate topolypropylene tubes (12 mnm×75 mmn), 2 ml of Trizol™ solution was addedand samples were homogenized with a Polytron homogenizer for 30 secondsat setting 10. The homogenized sample was transferred to a 14 ml sterilepolypropylene Falcon™ tube and incubated for 5 min at room temperature.To each sample, 400 μL of chloroform was added, and the tubes vigorouslyhand shaken for 15 sec and incubated for another 2-5 min at roomtemperature. Next, samples were centrifuged at 12,000×g for 15 min at 4°C. The aqueous phase was transferred to a fresh eppendorf tube, and 1 mlisopropanol was added to the tubes. The tubes were then vortexed, andthe RNA precipitated overnight at −20° C. Samples were centrifuged at10,000-12,000×g for 10 min at 4° C., the supernatant was removed, andthe pellet was washed two times with 75% ethanol (at least 1 ml). Thesample was mixed by vortexing and pelleted by centrifuging at 7,500×gfor 10 min at 4° C. The RNA pellet was briefly allowed to air dry (nomore than 10 min). The RNA pellet was dissolved in RNAse free water(50-100 μL per 100 mg of tissue) by gentle vortexing, incubated for 5-10min at 55-60° C. and stored at 70° C. Quantity and purity of RNA weredetermined by ultraviolet spectrophotometry at 260, 280 and 230 nm.

[0048] Northern Blot Analysis. Messenger RNA was measured by northernblot analysis as described by Zhao et al, Intern. J. Bioch. 25:1897-1903(1993). Aliquots of 15 μg total RNA were each dissolved in 10 μL loadinggel buffer (50% deionized formamide (vol/vol), 2M formaldehyde, 1.3%glycerol (vol/vol), 0.02M morpholinopropanesulphonic acid (MOPS), 5 mMsodium acetate, 1 mM EDTA and 0.1% bromophenol blue (wt/vol)). Thedissolved RNA aliquots were boiled for 2 min to denature the RNA, andthen loaded onto a 1% agarose (wt/vol) gel containing (0.66M)formaldehyde RNA was fractionated according to size by electrophoresisin the presence of a recirculating running buffer containing 0.02M MOPS,5 mM sodium acetate and 1 mM EDTA (5 hours at 100V). Afterelectrophoresis, the gels were soaked in two changes of 10×standardsaline citrate (SSC) (1.5M NaCl, 0.15M trisodium citrate, pH 7.0) andblotted onto a zeta-probe GT Genomi tested blotting membrane (BioRad,Mississauga, ON). The RNA was fixed onto membranes by baking in vacuumat 80° C. for 2 hours. Prior to hybridization with (³²P) CTP-Iabeledriboprobe, each membrane was prehybridized for 2 hours at 50° C. in 20ml of prehybridization buffer (deionized formamide (60% vol/vol),20×SSPE (5% vol/vol), 10% blotto (5% vol/vol), 20% SDS (5% vol/vol), and10 mg/ml sheared salmon DNA (denatured by boiling in a hot water bathfor 10 min, 5% vol/vol)). Hybridization was carried out for 12-16 hoursat 50° C. in an identical volume of fresh hybridization solution(deionized formamide (55% vol/vol), 20×SSPE (5% vol/vol), 10% blotto (5%vol/vol), 20% SDS (5% vol/vol), and 10 mg/ml sheared salmon DNA (2.5%vol/vol mixed with an equal part of deionized formamide. To this, 16.7KBq (1×10⁶ cpm) of labeled riboprobe was added and pre-warmed in a 70°C. water bath for 5 min before being added to the pre-warmedhybridization solution. The membranes were washed with 2×SSC at roomtemperature for 5 min and then in 2×SSC/0.1% SDS for either 10 min(GLUT2) or 15 min (proglucagon, SGLT-I). The membranes were transferredto a bath of 0.2×SSC/1% SDS as follows: proglucagon (70° C. for 10 min),SGLT-I (70° C. for 20 mins), and GLUT2 (60° C. for 2-3 min). Lastly, themembranes were washed in 0.2×SSC at room temperature for 2-3 min.Membranes were heat sealed in plastic bags and exposed to Kodak XRA5film (Eastman Kodak, Rochester, N.Y.) at −70° C. using an intensifyingscreen (Dupont Canada, Mississauga, ON). For statistical analysis, thesignals were quantified using laser densitometry (Model GS-670 ImagingDensitometer, BioRad Laboratories (Canada) LTD., Mississauga, ON). The28S and 18S ribosomal bands were quantified from negatives ofphotographs of the membranes. These bands were used to confirm theintegrity of the RNA and compensate for minor loading discrepancies.

[0049] Riboprobes. A 3.8 kb radiolabeled GLUT2 antisense riboprobe wasgenerated from Xba I-linearized plasmid DNA [pGEM4Z-HTL-3] and T7polymerase. The 350 kb proglucagon sense riboprobe was generated fromRsa 1-linearized plasmid DNA [pGEM4Z-HTL-3] and Sp6 polymerase. Lastly,the 2.1 kb SGLT-1 antisense riboprobe was generated from a 1.4 Kbfragment of lamb intestinal SGLT-1 clone (aa 207-664) (Wood et al,Bioch. Soc. Trans. 22:266s 1994 ).

[0050] BBM and BLM Isolation, Preparation and Enrichment. All procedureswere performed on ice using previously described procedures (Maenz andCheeseman Biochem. Biophsy. Acta 860(2):277-285 (1986)). Approximately 5gm of mucosal scrapings were added to 15 ml of membrane suspensionsolution, (MSS buffer 125 mM/I sucrose, 1 mM/I Tris-HCL, 0.05 mM/L PMSF,pH 7.4) and homogenized with a Polytron homogenizer for 30 seconds atsetting 8. Aliquots of this homogenate were then taken for enrichmentassays. The samples were split into two 30 ml eppendorf tubes and 20 mlof MSS buffer added to each tube. Each tube was homogenized twice moreat setting of 8 for 30 seconds. Samples were then centrifuged for 15 minat 2400×g, the supernatant was collected and centrifuged at 43,700×g for20 min. The remaining pellet consisted of two fractions. The outer whitefluffy layer comprised the basolateral membranes (BLM), and the innerdark brown pellet comprised the brush border membranes (BBM). BLM weregently resuspended in a small amount of MSS buffer and transferred to a14 ml eppendorf tube. BBM were resuspended in MSS buffer and samplesfrom the same animal were pooled into 1 tube and made up in 20 ml of MSSbuffer. BBM were then centrifuged for 20 min at 43,700×g. Again thefluffy white pellet was gently resuspended with MSS buffer and added tothe 14 ml eppendorf tube and the dark pellet was resuspended in exactly30 ml of MSS buffer. Isolated BLM were homogenized for 15 seconds onsetting 8. Each sample was loaded on 25 ml 20% Percoll® and centrifugedfor 30 min at 46,000×g. The resulting fluffy band in the Percollcollected and transferred to 25 mm×89 mm polycarbonate ultracentrifugetubes (Beckman Instruments Inc., Palo Alto, Calif.), then brought up tovolume (approximately 38 ml) with MSS buffer, and centrifuged at115,000×g for 30 min. The membrane layer was removed, diluted with 20 mlof MSS buffer, and homogenized for 15 seconds with the Polytron® atsetting 8. CaCl₂ (1M, 100 μL) was added and stirred gently on ice for 10min. Samples were centrifuged for 10 min at 7700×g, the pelletresuspended in 20 ml MSS buffer, and homogenized for 15 seconds atsetting 8. Samples were centrifuged another 20 min at 46,000×g and thepellet was resuspended in 1 ml MSS buffer. Aliquots were then taken formarker enrichment assays. BBM samples were homogenized for 15 secondswith the Polytron at setting 8 and centrifuged for 10 min at 1,900×g.The supernatants were transferred to new tubes and centrifuged another15 min at 14,600×g. Again, the supernatants were transferred to newtubes containing 300 μL of 1M CaCl₂, and stirred gently on ice for 20min. Samples were centrifuged for 30 min at 3,000×g, the supernatant wascollected, and centrifuged another 30 min at 46,000×g. The pellet wasresuspended in 1 ml of ddH₂O and aliquots were taken for enrichmentassays. The enrichment assay described by Esmann, Methods in Enzymology156:72-79 (1988) was used for the BLM enzyme Na⁺K⁺-ATPase. Total ATPaseactivity was assayed by incubating mucosal homogenates and membranepreparations in the presence of ATP and Mg²⁺, and measuring theliberated inorganic phosphate using the classic molybdenum reaction.Ouabain-insensitive ATPase activity was assayed as described above inthe presence of ouabain. Na⁺K⁺-ATPase activity is ouabain sensitive,therefore the difference between total and ouabain-insensitive ATPaseactivity is the Na⁺K⁺-ATPase activity. Results are expressed aspercent-fold enrichment. The enrichment assay for the BBM enzymealkaline phosphatase was measured using the alkaline phosphatase kitfrom Sigma (Cat #245-10, Sigma Diagnostics, St. Louis, Mo.). Theprocedure is based on the hydrolysis of p-nitrophenyl phosphate top-nitrophenol and inorganic phosphate by alkaline phosphatase. Thep-nitrophenol formed is yellow in color and shows a maximum absorbanceat 405 nm.

[0051] Western Blot Analysis. The western blot analysis protocoldescribed by Tappenden, PhD Thesis, University of Alberta, Edmonton,Canada (1997) was used for the quantification of BBM and BLM glucosetransporters. BLM (60 μg isolated protein) samples were diluted 1:4 with1×sample buffer (0.5M Tris-HCl pH 6.8 (13.2% vol/vol), glycerol (10.5%vol/vol), 0.05% (w/vol) bromophenol blue and 10% SDS (0.21% w/vol)). BBM(60 μg isolated protein) samples were diluted 3:1 with 4×sample buffer(0.24M Tris-HCL, 40% glycerol, 8% vol/vol of 10% w/vol SDS, 0.5 mLbromophenol blue). BBM samples were boiled for 10 min, but not the BLMsamples. The stacking gel (4.1M acrylamide/21 mM N′N-bis methyleneacryl(10.7% vol/vol), 0.5M Tris-HCL, pH 6.8 (0.24% vol/vol), 10% (w/vol) SDS(0.97% vol/vol), 10% APS w/v (4.86% vol/vol) and 0.4% TEMED (vol/vol))was placed on top of the separating gel (4.1M acrylamide/21 mM N′N-bismethyleneacryl (32.1% vol/vol), 1.5M Tris-HCL, pH 8.8 (32.1% vol/vol),10% (w/vol) SDS (1.3% vol/vol), 10% (w/vol) APS (0.66% vol/vol) and0.16% (vol/vol) TEMED). Electrophoresis was carried out in runningbuffer (0.3% Tris (w/vol), 1.44% glycine (w/vol) and 0.1% SDS)) at100-200 V for 1-2 hours until the dye front reached the end of the gel.Proteins were then transferred for 1.5-2 hours at 200 V onto anitrocellulose membrane (MSI Laboratories, Houston, Tex.) using atransfer unit (BioRad, Mississauga, ON) with transfer buffer (Tris-base(0.189% w/vol), glycine (0.9% w/vol), methanol (20% vol/vol), SDS (0.02%w/vol)). Following the transfer, the membranes were placed immediatelyinto TBST (1M Tris pH 7.5 (2% vol/vol), NaCl (0.88% w/vol), 0.05%Tween-20 (0.05% vol/vol)). Membranes were blocked in TBSTM (TBST with 5%(w/vol) powdered milk) for at least 1 hour with gentle agitation, andthen incubated with primary antibodies to SGLT-1 (Cat # AB1352,Chernicon International Inc., Temecula, Calif.) at a dilution of 1:1000or GLUT2 (Cat # AB1342) at a dilution of 1:500 overnight at 4° C.Membranes were washed 3×10 min in TBST with gentle agitation, and thenincubated with the secondary antibody (anti-rabbit IgG HRP-conjugate,Signal Transduction, PDI Bioscience, Inc., Aurora, ON) at a dilution of1:4000 for at least 2 hours with gentle agitation. Blots were coveredcompletely with Supersignal CL-HRP™ (Cat #34080, Pierce, Rockford, Ill.)working solution and incubated for 5 min before being exposed to KODAKXRA5 film. Loading consistency and protein transfer was confirmed bystaining the blots with Ponceau S (0.1% w/vol Ponceau S (BDH), 5% aceticacid). Statistical analysis was performed on the relative intensities ofthe bands. For statistical analysis, the signals were quantified usinglaser densitometry.

[0052] Measurement of Transport Kinetics. Transport kinetics weremeasured as described by Thomson and Rajotte, Am. J. Clin. Nutr.38:394-403 (1983). A 12 cm segment of intestine was removed from eachanimal, opened along the mesenteric border and carefully washed withice-cold saline to remove visible mucus and debris. Pieces of intestine(1 cm²) were cut out and the tissue was mounted as flat sheets inincubation chambers containing oxygenated Kreb's bicarbonate buffer (pH7.4) at 37° C. Tissue discs were preincubated in this buffer for 15 minto allow equilibration at this temperature. After preincubation, thechambers were transferred to beakers containing [³H] insulin and various[¹⁴C]-probe molecules in oxygenated Kreb's bicarbonate buffer (pH 7.4)at 37° C. The concentration of solutes was 4, 8, 16, 32 and 64 mM forD-glucose and D-fructose, 16 m for L-glucose, and 0.1 mM for lauricacid. The preincubation and incubation solutions were mixed usingcircular magnetic bars which were adjusted with a strobe light toachieve a stirring rate of 600 rpm and a low effective resistance of theintestinal unstirred water layer. The experiment was terminated byremoving the chambers, quickly rinsing the tissue in cold saline forapproximately 5 seconds and cutting the exposed mucosal tissue from thechamber with a circular steel punch. The tissue was dried overnight inan oven at 55° C. to determine the dry weight of the tissue and thensaponified with 0.75 N NaOH. Scintillation fluid (Beckman Ready Solv HP,Toronto, ON) was added to the sample and radioactivity determined usingan external standardization technique to correct for variable quenchingof the two isotopes (Beckman Beta LS-5801, Beckman Instruments Inc,Mountain View, Calif.). The uptake of nutrients was expressed asnmol/100 mg dry tissue/minute.

[0053] Villi Height and Crypt Depth Measurements. Intestinal segmentswere cut into sections. Intestinal villi height and crypt depths weremeasured under a light microscope using Northern Exposure Image Analysissoftware (Empix Imaging Inc., Mississauga, ON). A total of 10 recordingswere made for each animal and each segment, with the average used forstatistical analysis.

[0054] Statistical Analyses. All statistical analyses were performedusing the Statistical Analysis System (SAS) statistical package (version6.10, SAS Institute, Cary, N.C.). For proglucagon and SGLT-1 mRNAabundance, and SGL T-1 and GLUT2 transporter abundance, data wasanalyzed using the general linear models procedure (proc GLM) andsignificant differences were identified by one-way ANOVA. The modelincluded diet, gel, period, pair and diet period. Both period and dietperiod were found to be non-significant and subsequently excluded. Villiheight, crypt depth and intestinal GLP-1 concentrations were analyzedusing proc GLM and the one-way ANOVA that included diet and pair. Againboth period and diet period were non-significant and excluded from themodel. Plasma AUC for GLP-1, insulin and glucose were analyzed usingpaired T-tests within proc GLM. Repeated measures ANOVA was used toanalyze for differences between animal weights. The effect of period offeeding was tested but not significant (p>0.05). Intestinal transportrates for D-glucose, L-glucose, D-fructose and fatty acid 12 wereanalyzed using paired T-tests within proc GLM. Data presented aremeans±SEM. Significant differences were identified when p<0.05.

[0055] Effect of diet on body weight. Energy requirements wereindividually calculated and dietary portions were adjusted accordinglysuch that dog weights did not differ by experimental diet (23.4±1.8 kg,22.9±1.8 kg, 23.5±1.8 kg for pre-experimental, HFF and LFF respectively)or by period (23.4±1.8 kg, 23.4±1.8 kg, 23.4±1.8 kg for pre-experimental[day 7], period 1 [day 21], and period 3 [day 35], respectively).

[0056] Effect of OGTT on plasma GLP-1, insulin and glucose. Plasma GLP-1concentrations were increased at 30 and 90 min for dogs when fed the HFFvs the LFF diets (see, FIG. 1A). Insulin concentrations were increasedat 90 min for dogs when fed the HFF vs the LFF diets (see, FIG. 1B).Dietary fiber type did not influence blood glucose concentrations at anytime points during the OGTT (see, FIG. 1C). The incremental area underthe curve was significantly higher for GLP-1 (see, FIG. 2A, 988±92 vs648±92 pmol/L*h, P≦0.05) and insulin (see, FIG. 2B 15781±1371 vs.11209±1371 pmol/L*hr, p<0.05) when dogs were fed the HFF vs LFF diets.The area under the curve for glucose was significantly lower for dogswhen fed the HFF vs LFF diets (219±22 mmol/L*hr vs 291±22 mmol/L*hr,p≦0.05, see, FIG. 2C). This demonstrates that the fermentable fiber dietincreases the amount of GLP-1 and improves glucose homeostasis in thetested animals.

[0057] Effect of diet on intestinal proglucagon and GLP-1 concentration.Ingestion of HFF vs LFF diets resulted in greater proglucagon mRNAabundance in the ileum (1.13±0.04 vs. 0.83±0.04 densitometer units) andthe colon (1.45±0.05 vs. 0.78±0.05 densitometer units) (see, FIG. 3).Proglucagon mRNA expression was not detected in the duodenum. GLP-1concentrations, were significantly greater in mucosal scrapings fromdogs fed the HFF vs LFF diets (41±4 pmol GLP-1 /mg protein vs. 25±4 pmolGLP-1 /mg protein). This demonstrates again that the fermentable fiberdiet increases GLP-1 concentrations in the tested animals.

[0058] Histology. Dietary effects on intestinal villi heights and cryptdepths are presented in FIG. 4. Duodenal villi heights tended to behigher in dogs fed the HFF diet compared to those fed the LFF diet(1505±83 vs 1294±83 μm, p=0.1) but there were no differences in duodenalcrypt depths (289±28 vs 262±28 um). Jejunal villi heights weresignificantly higher in dogs fed the HFF vs LFF diets (1517±43 vs1343±43 μm, respectively) but no significant differences were found incrypt depths (277±19 vs 234±19 μm). Ileal villi heights and crypt depthswere not significantly different between dogs fed the HFF vs. LFF diet(1035±45 vs 993±45 μm and 251±46 vs 357±46 μm, respectively). Coloniccrypt depths were not significantly different (724±33 vs 727±33 μm)between dogs fed the HFF vs LFF diet, respectively.

[0059] Nutrient uptake. The effect of dietary fiber fermentability onnutrient uptake is shown in Table 2. Consumption of HFF resulted in asignificantly higher maximum glucose uptake capacity (Vmax) forD-glucose in the jejunum (see, FIG. 5). A significant diet effect wasalso noted in fatty acid-12 uptake in the jejunum, a measure ofunstirred water layer resistance. The Michaelis affinity constant (Km)was not affected by diet. The estimation of paracellular D-glucoseuptake, or the Kd for D-glucose as determined by extrapolation ofL-glucose uptakes at 16 mM through the origin and normalizing to 1 mM,was not significantly affected by diet. Kd for D-fructose was notaffected by diet.

[0060] Glucose Transporters. Diet did not affect SGLT-1 mRNA in any ofthe intestinal segments measured (see, FIG. 6). The consumption of HFFvs. LFF diet was associated with higher jejunal SGLT-1 transporterprotein abundance (22.2±3.7 vs 6.6±3.7 densitometer units). SGLT-1transporter protein abundance tended to be higher in the ileum when HFFdiet was consumed (13.4±0.7 vs 10.4±0.7 densitometer units, p=0.09, see,FIG. 7). Significant differences due to diet were seen in both jejunaland ileal GLUT2 transporter protein abundance (see, FIG. 8), showing anincrease with consumption of HFF vs. LFF diet (1.9±0.2 vs. 0.9±0.1densitometer units and 4.2±0.2 vs. 1.5±0.2 densitometer units,respectively). TABLE 1 Composition of experimental diets Low-FermentableHigh Fermentable Fiber Fiber (LFF) (HFF) Ingredient (g/kg diet as fed)poultry by-product meal 460 460 poultry fat 164 164 fishmeal 122 121pre-gelled cornstarch 80 110 Menhaden oil 3 3 dried whole egg 40 40Chicken digest 25 25 vitamin premix 3.2 3.2 mineral premix 2.4 2.4cellulose 70 — beet pulp — 60 gum arabic — 20 fructooligosaccharides —15 Potassium chloride 2.2 2.1 Calcium chloride 1.9 1.1 Choline chloride1.1 — Sodium chloride 0.3 0.3

[0061] TABLE 2 Intestinal transport rates in dogs fed highly-fermentablefiber (HFF) versus lowly-fermentable fiber (LFF) diets for 14 daysJejunum Ileum HFF LFF HFF LFF D-glucose¹ Vmax (nmol/mg tissue/min  182 ±15a  133 ± 13b  132 ± 11  146 ± 15 Km (mM) 10.0 ± 1.9  8.0 ± 2.0  5.5 ±1.2 12.7 ± 2.2 L-glucose (nmol/mg tissue/min) at 16 mM 21.7 ± 1.2 21.5 ±3.3 33.7 ± 5.3 27.8 ± 3.5 at 1 mM  1.4 ± 0.1  1.4 ± 0.2  2.1 ± 0.3  1.4± 0.2 D-fructose (nmol/mg tissue/min) Kd² 1.96 1.61 2.43 2.28 Fatty acid12 uptake³ (nmol/mg tissue/min)  2.4 ± 0.2a  1.7 ± 0.2^(b)  3.6 ± 0.5 4.2 ± 0.2

EXAMPLE 2

[0062] Two groups of five adult beagles each with both sexes, were fedtwo diets that differed only in the source of fiber (see Table 3).Cellulose, which is minimally degraded during passage through the caninegastrointestinal tract (GIT), was added to the control diet (A) at alevel of 3.6%. The second diet (B) contained beet pulp (4.2%) andfructooligosaccharides (FOS) (1%), which are fermented by the GITbacteria of dogs. Chemical analyses showed both diets had 25.9% protein,11.8% fat, with 6.2% moisture, 5.7% ash, 1.23% calcium, and 79%phosphorus. Diet B used a blend of beet pulp and FOS because differencesin their rates of fiber fermentation by the intestinal bacteria of dogs.The products of bacterial metabolism of FOS, such as SCFA, should beavailable more proximally in the GIT compared to those from beet pulp,which is fermented slower. Furthermore, the two sources of fermentablefiber are designed to yield different concentrations and proportions ofSCFA. The cellulose (Solka Floc) was obtained from Fiber and SalesDevelopment Corporation (St. Louis, Mo.), the beet pulp from MichiganSugar (Saginaw, Mich.), and FOS from Golden Technologies Company(Golden, Colo.). TABLE 3 Ingredient Portion of Diet, wt % corn grits to100 chicken and chicken by-product meal 23.4  brewers rice 15.9  chickenfat 4.2 fiber source a fish meal 3.3 vitamin and mineral premix 3.2chicken digest 2.0 dried egg product 1.4 fish oil  0.75 brewers driedyeast  0.47 flax  0.28 DL-methionine  0.19

[0063] The dogs were housed in two groups in separate open kennels. Thediets were fed for at least six weeks before surgery was performed.Immediately after surgery the small intestine was removed and theassociated mesenteries were severed so the intestine could bestraightened on a horizontal surface and length measured in a restingstate. Three segments of 25-30 cm in length were removed and immediatelyplaced in cold (2-4° C.) Ringers that had been aerated with a mixture ofO₂ and CO₂ (95% and 5%). The first segment originated from 30 cm distalto the pylorus and was considered as proximal intestine. The second wastaken from the mid point of the small intestine and was designated asmid intestine. The third segment, which started 30 cm from theileocolonic junction and proceeded proximally, was considered to berepresentative of distal intestine.

[0064] From each of the three segments of small intestine a 10 cm lengthwas used to determine wet weight per cm, circumference, and percentageof mucosa on a dry matter basis. Regional wet weights and nominalsurface area (not accounting tor area amplification by villi andmicrovilli) were estimated as the products of regional weight per cm andcircumference times regional length. Regional mucosal mass was estimatedby multiplying percent mucosa times regional wet weight. Values for theentire intestine were calculated by summing the three regions.

[0065] A modification of the everted sleeve method (Karasov et al, J.Comp. Physiol. B 152:105-116 (1983)) was used to measure rates ofnutrient uptake. Because adult beagles have a large diameter smallintestine (>1 cm), it was not practical to use entire sleeves to measurenutrient absorption. Instead, pieces of tissue of about 0.5 cm² weresecured by silk ligatures onto the sides and near the ends of 5 mm rodswith the mucosa exposed. Preliminary validation studies showed thatrates of uptake were comparable to those measured using intact sleevesof intestine (values differed <10% between mounting techniques). Thetissues were kept in cold, aerated Ringers before, during, and aftermounting onto the rods.

[0066] Measurements of uptake were performed at 37° C. and were started45 min after removal of the intestine. Following the protocol of Puchaland Buddington, Am. J. Physiol., 262:G895-902 (1992), the incubationsolutions consisted of Ringers with either glucose or proline.Accumulation of nutrient by the tissues was quantified by adding labeledL-proline (³H) or D-glucose (¹⁴C). Proline was selected as arepresentative amino acid since it has a “private” carrier (the iminoacid transporter), whereas other amino acid carriers can transportseveral different classes of amino acids with varying affinities.Polyethylene glycol (¹⁴C) was added to proline solutions to correct forproline associated with the adherent fluids and not actually absorbed.For glucose solutions, the passively absorbed isomer L-glucose (³H) wasadded, allowing for simultaneous correction of D-glucose present inadherent fluids and passively absorbed independent of carriers. Afterthe tissues were exposed to the nutrient solutions, they were removedfrom the rods (tissues exposed to glucose were first rinsed for 20seconds in cold Ringers), and placed in tared vials. After wet weightswere recorded, the tissues were solubilized, scintillant added, andassociated radioactivity was measured by liquid scintillation counting.Rates of glucose and proline uptake were calculated and expressed asfunctions of tissue weights.

[0067] The regional distribution of uptake was determined by incubatingtissues from each segment in solutions containing 50 mmol/L solutions ofglucose or proline. Preliminary studies showed that this concentrationis sufficiently high that it saturates the carriers and yields maximalrates of absorption. The maximum capacity of the entire length of smallintestine to absorb glucose and proline was estimated by summing theproducts of regional rates of uptake times regional wet weights.

[0068] Kinetics of uptake were defined in the proximal intestine forglucose and the mid intestine for proline. This was accomplished byexposing tissues to Ringers with 0.4, 0.2, 1, 5, 25, and 50 mmol/L ofunlabeled glucose and proline. Resulting uptake values were examined bynon-linear regression analysis to calculate maximal rates of uptake(Vmax) and apparent affinity constants (Km). For analysis of prolinedata, a passive permeation coefficient was included to account forproline absorbed passively and independent of carriers.

[0069] Values presented in tables and figures are means and standarderrors. ANOVA was used to search tor effects of diet and region ondimensions and rates of glucose and proline absorption. When asignificant regional effect was detected, Duncan's test was used toidentify specific differences. Analyses of the data were performed usingthe Statistical Analysis System (SAS, Version 6.LL, Cary, N.C.), withp<0.05 accepted as the critical level of significance.

[0070] Body weights did not differ between dogs fed the two diets.However, dogs fed the diet with the blend of beet pulp and FOS assources of fermentable fiber (Diet B) had intestines that were 22%longer than those fed the diet with non fermentable fiber (Diet A)(p=0.09; see Table 4). Circumference declined from proximal to distal(p<0.05). Although values did not differ significantly betweentreatments in any region, when all three regions were combined, dogs feddiet B with fermentable fiber had 28% more total intestinal surface areaavailable for absorption.

[0071] Wet weight per cm declined from proximal to distal in bothgroups, with significant differences between all regions (p<0.05).Intestines of dogs fed fermentable fiber had a higher average wet weightper cm (1.17 vs 1.04; p<0.05), due mainly to the greater mass of theproximal intestine (p<0.05). The wet weight per cm for mid and distalregions did not differ significantly between treatments. The highertotal intestinal wet weight of dogs fed fermentable fiber is thereforedue to a combination of having longer intestines with more weight per cmin the proximal intestine. Dry weight per cm also declined from proximalto distal (p<0.05), but did not differ between treatments in any region,indicating the heavier proximal intestine per cm of dogs fed fermentablefiber is partly due to higher water content. Even so, dogs fedfermentable fiber had more total intestinal dry mass.

[0072] The percentage of mucosa did not differ between regions (p>0.50)or between treatments in any region (p's>0.70). The averages for allthree regions were, respectively, 39%±0.02 and 39%±0.03 for dogs fed thediets with and without fermentable fiber. Because of greater totalintestinal weight, total intestinal mucosa mass was greater in dogs fedthe diet with fermentable fiber. TABLE 4 Parameter Diet A Diet B Bodyweight (kg) 10.18 ± 1.05 10.18 ± 0.47 Intestinal length (cm)   306 ± 26*  372 ± 23 Intestinal surface area (cm²)  1240 ± 95*  1582 ± 96Intestinal wet weight (g)   318 ± 23*   430 ± 17 Intestinal dry weight(g)  60.9 ± 3.1*  77.9 ± 5.7 % Mucosa   39 ± 3   39 ± 2

[0073] Values tor glucose uptake represent carrier mediated transport.Rates of uptake at the saturating concentration of 50 mmol/L werehighest in the proximal intestine (FIG. 9, p<0.05 for region effects),but were not significantly higher in dogs fed the diet with fermentablefiber (p>0.20 for treatment effects). Values for mid and distalintestine did not differ significantly from each other or betweentreatments.

[0074] Kinetic analysis of uptake by proximal intestine as a function ofglucose concentration (FIG. 10) showed saturable uptake for dogs fromboth treatments. Although values for uptake at 50 mmol/L did not differsignificantly between treatments, the kinetic analysis showed that dogsfed the diet with fermentable fiber had higher maximum rates of uptake(1.21±0.11 nmol/mg-min vs 0.60±0.13, p<0.05). Apparent affinityconstants did not differ between treatments (6.2±2.1 vs 3.9±3.3)implying only one transporter type was present.

[0075] Values for rates of proline uptake represent the sum ofcarrier-mediated uptake and passive, carrier-independent absorption.Values at 50 mmol/L did not differ between treatments or region (FIG.9).

[0076] Proline uptake by dogs fed the diet with cellulose (Diet A)increased monotonically with proline concentration (FIG. 11), did notshow any evidence of saturation kinetics typical of carrier-mediatedprocesses, and were best fit by a linear relationship. As an additionalindicator, ratios for the accumulation of tracer proline were calculatedat 0.04 mmol/L relative to 50 mmol/L. If carriers are present in limitednumbers, the labeled and unlabeled proline would have to compete forcarrier sites. This would result in ratios would that would exceed 1.0because of the reciprocal relationship between accumulation of tracerand nutrient concentration. Ratios for dogs fed the diet with celluloseaveraged 0.96±0.11 indicating a lack of competition. These findings, inconjunction with those from the kinetic analysis, indicate that passiveinflux represented nearly 100% total proline absorption and that therewere few transporters present or functioning.

[0077] In contrast, when dogs were fed the diet with fermentable fiber(Diet B), the relationship between rates of uptake and prolineconcentration deviated from linearity and was best fit by an equationthat included a saturable process and passive influx. This wascorroborated by tracer accumulation ratios that averaged 1.21±0.15. Thisvalue does not differ significantly from 1.0 and is markedly less thanratios for glucose (9.13±1.36 and 4.58±0.80 tor dogs fed diets with andwithout fermentable fibers, respectively, p<0.05 for comparisons with1.0 and between treatments). However, it is suggestive that morecarriers for proline are present in the mid intestine when dogs are feda diet with fermentable fiber. Even so, passive influx at 50 mmol/Lproline still contributes over 90% of total influx.

[0078] The affinity constant for proline uptake by intact tissues fromother vertebrates ranges from about 1 to 5 mmol/L. If the iminotransporter of dogs is similar to those known for other mammals, thenall of the carriers should be saturated at the concentration of 25mmol/L. Therefore, any increase in proline absorption between 25 and 50mmol/L should reflect passive, carrier-independent influx. When theslopes of the lines between 25 and 50 mmol/L were compared, nodifference between dogs fed diets A (0.074±0.022 nmol/mg-min-mmol/L) andB (0.054±0.011, p>0.20) could be detected.

[0079] When rates of uptake were integrated with regional wet weights,dogs fed the diet with fermentable fiber had higher total intestinalcapacities to absorb glucose (271±42 μmol/min vs 139±37; p<0.05). Thiswas mainly caused by higher values in proximal intestine; values for midand distal intestine did differ between treatments (FIG. 12). Treatmenteffects were not detected for proline uptake capacities in any region(FIG. 12), or for the entire length of small intestine (Diet A=1246±155,Diet B=1031±124; p>0.40).

[0080] This experiment demonstrates that the intestinal structure andfunctions of dogs are altered by the types of fibers present in thediet. The results demonstrate longer, heavier intestines with moresurface area and mucosa result when dogs are fed a diet with fibers thatcan be readily fermented by the GIT bacteria. The responses were morepronounced in the proximal intestine, as evident from the differences inproximal weight and mucosal mass between dogs fed the two diets. Thelack of difference in the percent of mucosa in proximal intestine, orany other region of the intestine (p>0.9) indicates there was anincrease in all tissue layers. However, because of greater mass of theproximal region, dogs fed the diet with fermentable fiber had moremucosa in the proximal region as well as the entire length of smallintestine. The implication is that dogs fed a diet with fermentablefibers have more intestine to hydrolyze and absorb dietary inputs. Theresults also indicate that including fermentable fibers in canine dietsprovide benefits to healthy dogs that are in addition to the increasesin the beneficial GIT bacteria.

[0081] While certain representative embodiments and details have beenshown for purposes of illustrating the invention, it will be apparent tothose skilled in the art that various changes in the methods andapparatus disclosed herein may be made without departing from the scopeof the invention, which is defined in the appended claims.

What is claimed is:
 1. A process for altering the function andcomposition of the gastrointestinal tract (GIT) of an animal to improveglucose metabolism comprising the steps of: feeding an animal a dietconsisting essentially of a composition containing fermentable fiberswhich have an organic matter disappearance of 15 to 60 percent whenfermented by fecal bacteria for a 24 hour period, said fibers beingpresent in amounts from about 1 to 11 eight percent of supplementaltotal dietary fiber, and maintaining said animal on said diet for asufficient period of time to allow said fermentable fibers to ferment inthe GIT of said animal.
 2. The process of claim 1 wherein saidcomposition contains from 2 to 10 weight percent of supplemental totaldietary fiber of said fermentable fibers.
 3. The process of claim 1wherein said composition contains from 3 to 9 weight percent ofsupplemental total dietary fiber of said fermentable fibers.
 4. Theprocess of claim 1 wherein said composition contains from 4 to 7 weightpercent of supplemental total dietary fiber of said fermentable fibers.5. The process of claim 1 wherein said fermentable fibers have anorganic matter disappearance of 20 to 50 percent.
 6. The process ofclaim 1 wherein said fermentable fibers have an organic matterdisappearance of 30 to 40 percent.
 7. The process of claim 1 whereinsaid fermentable fibers are selected from the group consisting of beetpulp, gum arabic, gum talha, psyllium, rice bran, carob bean gum, citruspulp, pectin, fructooligosaccharides, mannanoligosaccharides andmixtures thereof.
 8. The process of claim 1 wherein said fermentablefibers are selected from the group consisting of beet pulp, gum arabicand fructooligosaccharides.
 9. The process of claim 1 wherein saidfermentable fibers comprise a blend of beet pulp, gum talha andfructooligosaccharides.
 10. The process of claim 9 wherein the ratio ofsaid beet pulp to said fructooligosaccharides in said blend is betweenabout 3:1 to about 6:1.
 11. The process of claim 9 wherein the ratio ofsaid beet pulp to said fructooligosaccharides in said blend is about4:1.
 12. The process of claim 9 wherein the ratio of said beet pulp togum talha to fructooligosaccharides is about 6:2:1.5.
 13. The process ofclaim 1 wherein said animal is a dog.
 14. A process for increasing thesecretion of glucagon-like peptide-1 (GLP-1) the gastrointestinal tract(GIT) of an animal to improve glucose metabolism and satiety in saidanimal comprising the steps of: feeding said animal a diet consistingessentially of a composition containing fermentable fibers which have anorganic matter disappearance of 15 to 60 percent when fermented by fecalbacteria for a 24 hour period, said fibers being present in amounts fromabout 1 to 11 weight percent of supplemental total dietary fiber, andmaintaining said animal on said diet for a sufficient period of time toallow said fermentable fibers to ferment in the GIT of said animal toincrease the secretion of GLP-1 in the gastrointestinal tract of saidanimal.
 15. A process for improving nutrient absorption in thegastrointestinal tract (GIT) of an animal comprising the steps of:feeding an animal a diet consisting essentially of a compositioncontaining fermentable fibers which have an organic matter disappearanceof 15 to 60 percent when fermented by fecal bacteria for a 24 hourperiod, said fibers being present in amounts from about to 1 to 11weight percent of supplemental total dietary fiber, and maintaining saidanimal on said diet for a sufficient period of time to allow saidfermentable fibers to ferment in the GIT of said animal to increase thetransport of D-glucose and lauric acid in the gastrointestinal tract ofsaid animal.
 16. A process for treating an animal suffering fromexocrine pancreatic insufficiency (EPI) comprising the steps of: feedingsaid animal a diet consisting essentially of a composition containingfermentable fibers which have an organic matter disappearance of 15 to60 percent when fermented by fecal bacteria for a 24 hour period, saidfibers being present in amounts from about 1 to 11 weight percent ofsupplemental total dietary fiber, and maintaining said animal on saiddiet for a sufficient period of time to allow said fermentable fibers toferment in the GIT of said animal to increase nutrient absorption andthe transport of D-glucose and lauric acid in the gastrointestinal tractof said animal.